Vitamin K functions in a post-translational microsomal carboxylation that converts peptidyl glutamic acid to gamma-carboxyglutamic acid. The reaction requires vitamin K, a source of reducing equivalents, and molecular oxygen. Coumarin and indandione anticoagulants affect the biosynthesis of prothrombin by inhibiting the carboxylation. During the carboxylation, the vitamin is converted to a 2,3-epoxide that can be converted back to the vitamin by the action of epoxide reductase. The interconversion of vitamin K and epoxide is inhibitied by coumarin anticoagulants in vivo. However, in vitro, the effect of coumarins on these reactions is variable. The research outline in this proposal is designed to determine the mechanism(s) whereby coumarin anticoagulants inhibit hepatic carboxylation and cyclic interconversion of the vitamin and epoxide. We specifically propose to develop coumarin-sensitive, soluble enzyme systems for the carboxylation of exogenous peptide substrates, epoxidation of vitamin K, and reduction of vitamin K epoxide from rat liver. These solubilized systems will be further purified by conventional and affinity chromatographic procedures to provide enzyme systems to study the mechanism of coumarin anticoagulant action. Enzyme isolated from normal rats will be compared to those from warfarin-resistant rats in order to determine the mechanism of resistance. Photo-affinity labeling techniques will be developed to provide labeled enzymes for use as radioactive markers during purification. These affinity-labeling substrates will also be used to study enzyme mechanisms and binding sites. High performance liquid chromatography (HPLC) will be used for the analysis of vitamin K, epoxides, and carboxylated peptides obtained from these incubations. Use of HPLC in the analysis of these enzymes will enable a variety of synthetic substrates to be examined, coupled with increased sensitivity and improved quantitation. These enzymes will also be studied in extrahepatic tissue in both normal and warfarin-resistant rats. The results of this study should help in understanding coumarin therapy.